A phase I clinical trial of replication-competent Ad4-HIVenv and Ad4-HIVmosaic gag vaccines was initiated in the NIH clinical center in collaboration with NIAID and Dr. Mark Connors as Principal Investigator. The study is evaluating the safety and immunogenicity of the vaccines formulated as enteric coated capsules for oral administration and as a liquid for administration to the upper respiratory tract. At the same time, new replication-competent Ad vectors are under development for future clinical use following pre-clinical evaluation in the rhesus macaque model. Studies in mice and pilot studies in rhesus macaques have shown that Ad-HIV envelope recombinants with deletions of E1B55K and/or E4orf1-4 or E4orf1 early region genes not only have greater carrying capacity but also exhibit enhanced cellular and humoral immunity. These pilot studies will identify the optimal vector for use in subsequent pre-clinical vaccine studies evaluating overall immunogenicity and protective efficacy. An on-going pre-clinical vaccine study in rhesus macaques has investigated combination mucosal-systemic vaccine regimens. Neither vaccine regimen induced delayed SIV acquisition in vaccinated macaques, but this was partly due to development of innate immune memory (so-called trained immunity). We are currently investigating this response further using RNA seq studies and flow cytometry investigation of monocyte/macrophages to strengthen the conclusion. Although delayed acquisition was not obtained, we did observe modestly decreased acute viremia attributed to the vaccinated female macaques rather than the males. We previously reported a sex bias in SIV vaccine induced protection, and this current finding confirms the observation. Here, we have found the difference between the sexes is largely due to rectal microbiome differences which are affected by the vaccine regimen and in turn influence immune responses. A current pre-clinical study in macaques is evaluating a novel vaccine/microbicide regimen. Microbicides have been shown to be effective in preventing HIV transmission, however, their effectiveness depends on appropriate use which is often compromised by human behavior. We hypothesized that combining a prophylactic vaccine with a microbicide might provide protection against infection in instances when microbicides are not used properly. Therefore, we immunized rhesus macaques with our Ad-recombinant priming/Env protein boosting regimen in order to elicit mucosal immunity and subsequently exposed the vaccinated animals to infectious SIV following administration of a microbicide. The microbicide (SAMT-247) is a zinc finger inhibitor that results in expression of non-infectious viral particles which nevertheless have intact envelope on their surface. We have postulated that the non-infectious particles will boost the immune response already elicited by the vaccine regimen. Overall, we are testing the hypothesis that the combined vaccine plus microbicide regimen is beneficial by providing either additive or synergistic protection against SIV infection. To date we have found that the microbicide is highly effective and improves the protection obtained with the vaccine only. Finally, we recently reported the development of a new technology for sorting and characterization of HIV/SIV from clinical specimens using nanoFACS. The virions can be stained and sorted based on either cellular antigens incorporated into the virion particles or alternatively using specific antibodies to different envelope epitopes. The ability to sort infectious HIV from clinically relevant samples provides material for detailed molecular, genetic, and proteomic analyses applicable to future design of vaccine antigens and potential development of personalized treatment regimens.